Pathogenesis of myocardial fibrosis in spontaneously hypertensive rats (SHR)

The pathomechanisms of myocardial fibrosis are incompletelyunderstood. Coronary microvessels (MV), interstitial reactionsand focal myocardial lesions characterized by morphologicalsigns of ischaemia were found in 39 spontaneous hypertensiverats (SHR) and 33 control rats aged 3, 13, 27, 52 and 78 weeks,using new morphological preparations and measuring methods.In the developing phase of spontaneous hypertension (SH) thenumerical and area densities (developed for up to 13 weeks)of MV were lower than at 3 weeks and lower than control. Thesame was true for the fibrotic tissue density. However, allvalues were increased in the manifest phase of SH (27–78weeks). The increase in interstitial tissue is topologicallyand causally related to pathological MV reactions that representmorphologically chronically increased contractions. Small MVthat mostly elude detection with conventional staining methodsare of particular importance. The first phase of manifest SH(27th–52nd week) is characterized by a generalized developmentof myocardial fibrosi the late phase (52nd–78th week)by a reinforced localized fibrosis that is attributable to theenhanced progression of focal ischaemic myocardial lesions.The septal region is not included in this phase. Thus, in thetwo phases of manifest SH, the pathomechanisms responsible forthe development of myocardial fibrosis must be different.     

Relaxin reverses cardiac and renal fibrosis in spontaneously hypertensive rats

The antifibrotic effects of the peptide hormone relaxin on cardiac and renal fibrosis were studied in 9- to 10-month-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Rats (n=8 to 9 per group) were allocated into 3 groups: WKY controls, vehicle-treated SHR (SHR-V), and relaxin-treated SHR (SHR-R). Relaxin (0.5 mg/kg per day) was administered via subcutaneously implanted osmotic mini-pumps over 2 weeks before hearts and kidneys were harvested for analysis. Collagen content was analyzed by hydroxyproline assay, gel electrophoresis, and quantitative histology. Zymography was used to determine matrix metalloproteinase (MMP) expression and Western blotting to determine proliferating cell nuclear antigen (PCNA) expression and alpha-smooth muscle actin (alpha-SMA)/myofibroblast expression, whereas cardiac hypertrophy was assessed by myocyte size and real-time polymerase chain reaction of associated genes. The left ventricular (LV) myocardium of SHR-V contained increased collagen levels (by 25+/-1%, P<0.01 using biochemical analysis and 3-fold; P<0.01 using quantitative histology), enhanced expression of PCNA (by 70+/-8%; P<0.01), alpha-SMA (by 32+/-2%; P<0.05), and the collagen-degrading enzyme MMP-9 (by 70+/-6%; P<0.05) versus respective levels measured in WKY controls. The kidneys of SHR-V also contained increased collagen (25+/-2%, P<0.05 using biochemical analysis and 2.4-fold; P<0.01 using quantitative histology). Relaxin treatment significantly normalized collagen content in the LV (P<0.01) and kidney (P<0.05), completely inhibited cell proliferation (P<0.01) and fibroblast differentiation (P<0.05) in the LV, and increased MMP-2 expression (by 25+/-1%; P<0.05) without affecting MMP-9 in the LV compared with that measured in SHR-V. Thus, relaxin is a potent antifibrotic hormone with a rapid-occurring efficacy that may have therapeutic potential for hypertensive disease.     

Changes in myocardial collagen in spontaneously hypertensive rats (SHR)]

The aim of this study was to evaluate whether left ventricular hypertrophy in spontaneously hypertensive rats (SHR) is accompanied by myocardial collagen quantitative and/or qualitative changes. 15 SHR and 20 control normotensive rats (WKR) of three months of age were used. At this age, hypertension has already caused a significant increase in the ratio of the ventricular mass to body weight (mg/g) in hypertensive animals (SHR: 5.11 +/- 0.21; WKR: 3.40 +/- 0.22; p less than 0.001). With respect to body mass, the amount of collagen elicited from the hydroxyproline concentration increases in SHR but remains percentually the same with respect to the biventricular mass. In SHR, changes in the amount of type-1 alpha chains and type-V alpha chains, and the presence of a low molecular weight collagenous fraction have been observed. Moreover, we have found an increase in the ratio of type-1 alpha 1 chains to type-1 alpha 2 chains. This change might be related to the appearance of a type-1 alpha 1 trymer. The presence of such a type-1 alpha trymer and of low molecular weight collagenous fractions may suggest the appearance of fetal collagenous isoforms in ventricular myocardium, due to the increased pressure load as well as to the increased turnover (an index of a remodelling activity of cardiac stroma). These changes might play a role in the transformation of myocardial viscoelastic properties in SHR with a progressive diastolic stiffness of the ventricular wall.     

Nebivolol prevents myocardial fibrosis and diastolic dysfunction in salt-loaded spontaneously hypertensive rats

BackgroundWe have demonstrated previously that a high-salt diet (HS) produces myocardial fibrosis, left ventricular (LV) dysfunction, and renal insufficiency in adult spontaneously hypertensive rats (SHR), and that blockade of the renin-angiotensin system prevented those adverse effects of HS.Methods and ResultsEight-week-old male SHR were divided into four groups: controls received regular rat chow (0.6 NaCl); the other three were given HS. The second group was given placebo; the third, nebivolol (2 × 10 mg/kg/day) orally; and, the fourth, the same dose of nebivolol by osmotic minipump. Rats received respective treatments for 8 weeks. The data demonstrated that the HS induced increased cardiac mass (2.85 ± 0.05 vs. 5.36 ± 0.22 mg/g; P < .05 in control and HS groups, respectively); LV fibrosis as indicated by higher hydroxyproline concentration; further increase in arterial pressure (161 ± 7 vs. 184 ± 8 mm Hg; P < .05); myocardial ischemia; and LV diastolic dysfunction. Nebivolol ameliorated the adverse cardiac effects of HS, demonstrated by decreased LV mass and fibrosis and improved coronary hemodynamics and LV function.ConclusionsThe effects of nebivolol were independent of arterial pressure. The results of this study provide important laboratory data that support a rationale for nebivolol in the treatment of patients with hypertension having diastolic dysfunction with preserved ejection fraction.     

自发高血压大鼠左心室心肌胶原含量与舒张功能的相关性研究

目的测定自发高血压大鼠(SHR)左心室心肌胶原含量和舒张功能的参数,探讨二者的相关性,并揭示它们的增龄性改变。方法雄性SHR共30只(8、14周和20周各8只,32周6只),行组织多普勒成像(TDI)检测左心室舒张功能。处死后取左心室心肌,用羟脯氨酸试剂盒测定羟脯氨酸含量;反转录-聚合酶链反应(RT-PCR)检测Ⅰ型和Ⅲ型胶原的mRNA表达。结果 TDI显示,二尖瓣环舒张早期速度(Ea)随增龄减低(P0.05),二尖瓣环舒张早期与晚期速度比值(Ea/Aa)32周比8周时减低(P0.01),左心室等容舒张时间(TVRT)随增龄延长(P0.05),Tei指数随增龄增加(P0.05)。心肌羟脯氨酸含量、Ⅰ型和Ⅲ型胶原的mRNA表达随增龄均增加(均为P0.05)。羟脯氨酸含量、Ⅰ型和Ⅲ型胶原mRNA表达与TDI指标Ea、Ea/Aa均早负相关(r=-0.713～-0.431,P0.001～0.05),与IVRT均呈正相关(r=0.427～0.721,P0.001～0.01),与Tei指数均呈正相关(r=0.413～0.576,P0.001～0.01)。结论 SHR左心室心肌胶原含量呈增龄性增加,舒张功能呈增龄性降低,二者具有相关性。     

肝细胞生长因子与自发性高血压大鼠心肌纤维化的相关研究

目的 探讨高血压进展过程中肝细胞生长因子(hepatocyte growth facor,HGF)表达与心肌纤维化的关系及血管紧张素Ⅱ受体1(ATl)拮抗剂洛沙坦的干预作用. 方法 以不同周龄自发性高血压大鼠(SHR)为心肌纤维化模型,WKY(Wistar-Kyoto)大鼠为对照组.用洛沙坦对SHR大鼠进行治疗.各周龄组WKY、SHR大鼠及干预后的SHR大鼠处死后取心脏制成石蜡切片,利用麦松三色法检测心肌胶原,免疫组织化学方法检测心肌组织HGF,Leica Qwin彩色图像分析系统分析图片. 结果 SHR大鼠心肌胶原容积(collagen volume fraction,CVF)随周龄增长而增加,8、12、16、24和32周分别为(1.8±0.1)%、(1.8±0.1)0A、(3.8±0.4)%、(7.3±0.4)%和(13.4±1.8)%,与心肌组织HGF含量呈负相关(r=-0.8820,P<0.05).洛沙坦十预可增加HGF表达,降低CVF.结论 高血压心肌纤维化可能与心肌组织HGF抗纤维化作用降低有关,AT<,1>拮抗剂治疗可以增加心肌组织HGF表达从而改善心肌纤维化.     

Angiotensin type 2 receptor agonist compound 21 reduces vascular injury and myocardial fibrosis in stroke-prone spontaneously hypertensive rats

-Angiotensin type 2 receptor-mediated effects of angiotensin II appear to counteract many of the effects mediated via the angiotensin type 1 receptor. Compound 21 (C21), a selective angiotensin type 2 receptor agonist, has demonstrated beneficial effects on cardiac function after myocardial infarction in rodents. We hypothesized that C21 alone or in combination with an angiotensin type 1 receptor antagonist would blunt the development of hypertension and vascular damage in stroke-prone spontaneously hypertensive rats. Six-week-old stroke-prone spontaneously hypertensive rats received C21 (1 mg/kg per day), the angiotensin type 1 receptor antagonist losartan (10 mg/kg per day), C21 plus losartan, or vehicle PO for 6 weeks. Systolic blood pressure was lower in losartan and C21-losartan combination groups (P<0.001). Endothelium-dependent relaxation was enhanced (P<0.001) in the C21-losartan combination group at lower acetylcholine concentrations. C21 or C21-losartan combination reduced vascular stiffness, aortic medial and myocardial interstitial collagen content, and aortic fibronectin (P<0.05). C21 and losartan decreased the expression of 2 genes associated with cardiac hypertrophy, myosin heavy chain-β (myh7) by 30 to 50%, and α-skeletal muscle actin by 30% to 35% (P<0.05). C21-losartan combination caused an additional 40% reduction in myh7 compared with C21 (P<0.01). Aortic superoxide generation was reduced equally by the 3 treatments (P<0.001). Monocyte/macrophage infiltration in the aorta and kidney (P<0.001) and T-lymphocyte infiltration in the renal cortex (P<0.05) were lowered similarly by the 3 treatments. These data suggest that C21 alone or in combination with losartan may improve endothelial function and vascular composition and mechanics by reducing oxidative stress, collagen content, fibronectin, and inflammatory cell infiltration in stroke-prone spontaneously hypertensive rats.     

福辛普利对SHR左室肥厚和心肌纤维化的影响

目的探讨福辛普利对自发性高血压大鼠(SHR)左心室肥厚(LVH)和心肌纤维化的影响。方法18只16周龄SHR大鼠,随机分为福辛普利治疗组(SHR-F)和SHR空白对照组(SHR-C);另设同源的WKY大鼠8只为正常对照组。治疗组口服福辛普利20mg·kg-1·d-1,给药8周后处死动物,取左心室心肌称重,计算左心室/体重比(LVW/BW),Masson三色法染色观察左心室心肌胶原变化,计算机图像分析测量心肌切片的胶原容积分数(CVF)和血管周围胶原面积(PVCA)。结果SHR空白对照组的SBP、LVW/BW、CVF、PVCA均显著高于WKY对照组(P<0.01),与SHR空白对照组相比,福新普利治疗组能有效降低SHR的SBP,改善SHR左心室肥厚(P<0.01〉并使左心室内膜及心肌小动脉周围的胶原减少(P<0.01〉。结论福辛普利可有效降低SHR血压,部分逆转心肌纤维化和左室肥厚。     

胰激肽原酶对自发性高血压大鼠氧化损伤和心肌纤维化的影响

目的探讨胰激肽原酶对自发性高血压大鼠(SHR)抗氧化损伤和心肌纤维化的影响。方法15周龄雄性SHR大鼠24只,随机分成SHR对照组、胰激肽原酶小剂量组、大剂量组,每组8只。另取8只WKY大鼠作为正常血压对照组。小剂量组(7.2 U.kg-1.d-1)和大剂量组(14.4 U.kg-1.d-1)给予胰激肽原酶腹腔注射。治疗4 w后,测量各组收缩压(SBP)、左心室重量指数(LVM I)、心肌胶原容积分数(CVF)和心肌血管周围胶原面积(PVCA),同时测定血清中超氧化物岐化酶(SOD)、丙二醛(MDA)含量。结果治疗组SBP、CVF、PVCA与SHR对照组比较明显降低(P<0.05),仍未达到WKY组水平(P<0.05)。治疗组血清中SOD含量明显上升,MDA含量下降,与WKY组差异无显著性。结论胰激肽原酶能降低血压,减轻过氧化损伤,逆转心肌纤维化。且无明显毒副作用,有望成为治疗高血压左室肥厚新的有效药物。     

AT1 receptor blockade reduces cardiac calcineurin activity in hypertensive rats

The possible role of calcineurin in the attenuation of cardiac hypertrophy and fibrosis by blockade of the angiotensin II type 1 (AT1) receptor was investigated in Dahl salt-sensitive (DS) rats. The effect of the calcineurin inhibitor FK506 was also studied. DS rats progressively developed severe hypertension when fed a diet containing 8% NaCl from 7 weeks of age. In addition, marked cardiac hypertrophy and fibrosis were apparent and the activity of calcineurin and its mRNA expression in the myocardium was increased in these animals at 12 weeks in comparison with age-matched Dahl salt-resistant rats. The abundance of angiotensin-converting enzyme (ACE) and transforming growth factor (TGF)-beta1 mRNAs was also increased in the hearts of DS rats at 12 weeks. Treatment of DS rats with a non-antihypertensive dose of the selective AT1 receptor blocker candesartan (1 mg/kg per day) or FK506 (0.1 mg/kg per day) from 7 to 12 weeks attenuated both calcineurin activity and its mRNA expression in the heart, as well as the development of cardiac hypertrophy and fibrosis, without affecting cardiac function. Treatment with candesartan, but not FK506, prevented the upregulation of ACE and TGF-beta1 gene expression. Both candesartan and FK506 prevented the load-induced induction of fetal-type cardiac genes. These results demonstrate that AT1 receptor blockade attenuates the development of cardiac hypertrophy and fibrosis as well as the activation of calcineurin, without an antihypertensive effect, in rats with salt-sensitive hypertension. Calcineurin may be downstream from TGF-beta1 in AT1 receptor-mediated angiotensin II signaling in vivo.     

Perturbation of D1 dopamine and AT1 receptor interaction in spontaneously hypertensive rats

The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10(-7) mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29+/-2 density units, DU) than in SHR RPT cells (21+/-2 DU, n=7 per group, P<0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10(-7) mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.     

花生四烯酸环氧化酶-2途径参与丹参改善老年自发性高血压大鼠的心肌纤维化

目的探讨丹参对于老年自发性高血压大鼠（SHR）心肌纤维化的逆转作用和可能的作用机制——花生四烯酸环氧化酶途径。方法实验采用17个月龄的SHR分别连续给予12周的丹参注射液和生理盐水对照,选取同周龄的WKY大鼠作为正常对照,同时选用花生四烯酸环氧化酶-2（COX-2）的特异性抑制剂塞来昔布灌胃处理后注射丹参。测定血压、血流动力学、左心室质量指数,HE染色和Masson染色分析心肌细胞的大小或心肌纤维化水平,左心室心肌COX-2的蛋白表达和活性水平。结果与老年WKY大鼠相比,老年SHR高血压对照组血压显著增高f收缩压：（171±12）vs（125-I-3）mmHg;舒张压：（115±9）VS（87±3）mmHg;平均动脉压：（139±10）vs（106±5）mmHg;P〈0．05],心功能恶化[LVEDP：（13．9±1．7）VS（7．6．4-1．3）mmHg;LVdP／dtmax：（2528±167）VS（3015±217）mmHg／s;-LVdP／dtmax：（1957±134）vs（2501±175）mmHg／s,P〈0．05],左室质量指数增加[（3．45±0．07）VS（2．23±0．06）mg／g,P〈0．051,心肌细胞增大[心肌细胞直径：（23．5±0．4）VS（14．3±0．4）仙ITI,P〈0．051,纤维化严重[心肌间质纤维化指数：（1．66±0．05）％vJ（0．64±0．05）％;心肌血管周围纤维化指数：（139±9）％w（68±7）％,P〈0．05],心肌COX-2的蛋白表达水平增加[（125．8±7．2）VS（47．6±3．8）,P〈0．05]以及活性增强[（73．9±5．6）vs（56．7±4．4）kU／g,P〈0．05]。应用丹参的SHR组,收缩压无显著变化,其他指标均有显著下降（P〈0．05）,塞来昔布能够部分消除丹参的作用。结论丹参能显著逆转老年SHR的心肌纤维化水平,该作用可能是通过花生四烯酸环氧化酶途径。     

Histological and histometrical study of myocardial fibrosis in spontaneously hypertensive rats of the stroke-prone strain

In order to obtain fundamental information about the developmental mechanisms of myocardial fibrosis in chronic hypertension, the hearts of male spontaneously hypertensive rats of the stroke-prone strain (SHRSP) and Wistar rats of the Kyoto strain (WKY) were histologically and histometrically examined. Fibrosis was a prominent histological feature of the hearts in SHRSP. In consisted of focal, interstitial, and perivascular fibrosis. For histometrical analysis the percentage areas of interstitial and perivascular fibrosis were calculated by using a color image processor. The percentage area of myocardial fibrosis increased with advancing age in both SHRSP and WKY. However, it was significantly higher in SHRSP than in WKY at 18 and 30 weeks of age. In SHRSP perivascular fibrosis of small arteries had already appeared at 8 weeks of age, while perivascular fibrosis of arterioles and interstitial fibrosis developed later. It is supposed that perivascular as well as interstitial fibrosis is induced by the exudation of some growth factors due to an increased vascular permeability. On the other hand, the focal fibrosis observed in old SHRSP is suspected to occur as a result of injury in myocardium due to stenosis or occlusion of vessels.     

吡格列酮对老年自发性高血压大鼠心肌纤维化及胶原代谢的影响

目的探讨吡格列酮对老年自发性高血压大鼠(SHRs)心肌纤维化及胶原代谢的影响和可能机制。方法 12月龄SHRs分为吡格列酮组和SHR组,同月龄WKY鼠为对照组。观察大鼠体重及尾动脉血压、测定左室重量指数(LVI)、心肌胶原定性分析、测定心肌羟脯氨酸含量、Westen印迹法检测心肌基质金属蛋白酶(MMP)-1、MMP抑制物(TIMP)-1蛋白表达、测定心肌活性氧(ROS)和超氧化物歧化酶(SOD)水平并观察心肌过氧化物酶体增殖物激活受体(PPAR)γmRNA表达和组织定位。结果与对照组相比,SHR组血压、胶原容积积分、血管周围胶原面积和左室心肌组织羟脯氨酸浓度明显升高,吡格列酮组能改善心肌纤维化;SHR组MMP-1/TIMP-1显著降低,吡格列酮组MMP-1/TIMP-1水平显著升高,PPARγ表达增多,心肌ROS浓度显著下降(P0.05)。结论吡格列酮通过调节MMPs/TIMP平衡促进胶原降解,抑制胶原的沉积,抑制心肌纤维化,机制可能是通过激活PPARγ和抑制ROS的形成。     

Glomerular dimensions in spontaneously hypertensive rats: effects of AT1 antagonism

OBJECTIVE: A reduction in glomerular number and/or size has been implicated in the development of hypertension. This study investigated whether differences in glomerular number and/or size occur during the development of hypertension in the spontaneously hypertensive rat (SHR) and whether angiotensin II is responsible for any glomerular differences. METHODS: SHR (n=6) and Wistar-Kyoto (WKY) rats (n=6) were administered the angiotensin II type I receptor antagonist TCV-116 from 4 to 10 weeks of age. At 10 weeks of age, the kidneys from these rats and those from untreated SHR (n=6) and WKY rats (n=6) controls were perfusion fixed at physiological pressures and analysed using unbiased stereological techniques. RESULTS: There were no significant differences in glomerular number, glomerular volume or total glomerular volume between SHR and WKY rats. Treatment of SHR with TCV-116 significantly lowered systolic blood pressure but had no significant effect on glomerular number or volume or total glomerular volume. Treatment of WKY rats with TCV-116 reduced systolic blood pressure, body weight, glomerular volume and total glomerular volume; however, total glomerular volume per body weight of treated WKY rats was not significantly different from that of untreated WKY rats. CONCLUSION: There were no differences in glomerular number or volume in SHR compared with WKY rats at 10 weeks of age. We therefore conclude that glomerular changes are not responsible for the development of hypertension in SHR. Angiotensin II, via the type 1 receptor, does not contribute to glomerular growth during the development of hypertension in the SHR.     

罗格列酮对SHR大鼠AT1受体和mRNA的影响

24只自发性高血压大鼠（SHR）随机分组，12只给予罗格列酮，12只为空白对照组。采用尾动脉血压间接测定法。用原位杂交和免疫组化检测AT1受体和mRNA表达，观察8周。罗格列酮组SHR大鼠血压明显降低，AT1受体和mRNA表达水平受到抑制。罗格列酮可能通过抑制AT1表达发挥降压和靶器官保护作用。